The present invention relates to novel intermediates in the synthesis of camptothecin and related compounds, and to methods of synthesis of camptothecin and related compounds via a 4+1 radical annulation.
As part of an antitumor screening program, Wall and coworkers identified the novel pyrrolo [3,4-b] quinoline alkaloid (S)-camptothecin in 1966. Wall, M. E., et al., J. Am. Chem. Soc. , 88, 3888 (1966); Carte, B. K., et al., Tetrahedron, 46, 2747 (1990). The chemical formula of (S)-camptothecin is provided below. 
This compound had been isolated from the extracts of the camptotheca acuminata tree. In addition to its novel structure, camptothecin has two other unusual features: its quinoline nitrogen is not very basic, and its xcex1-hydroxy lactone is quite reactive. For a few years, camptothecin appeared to be an exciting lead compound for cancer chemotherapy. However, initial medical excitement waned because of the relative insolubility of camptothecin. Moreover, clinical trials of a water-soluble sodium salt derived by opening the lactone of camptothecin were abandoned because of unpredictable toxicity problems. The sodium salt is considerably less potent than camptothecin and its activity is now thought to result from lactonization to reform camptothecin in vivo. These observations delayed preclinical and clinical research of camptothecin and its analogs for 20 years.
However, oncological and medicinal interest in camptothecin was reborn in the mid 80s when details about the unique mechanism of action of camptothecin and its analogs began to unfold. Camptothecin acts on DNA through the intermediacy of the enzyme topoisomerase I. See Kaufman, S. H., et al., J. Nat""l. Cancer Inst, 85, 271 (1993); Hsiang, Y. H., et al., J. Biol. Chem. 260, 14873 (1985); Hsiang, Y. H. and Liu, L. F., Cancer Res., 48, 1722 (1988); Liu, L. F., Annu. Rev. Biochem., 58, 351 (1989); xe2x80x9cChemotherapy: Topoisomerases as Targets,xe2x80x9d Lancet, 335, 82 (1990). The topoisomerases solve topological problems of DNA. Human topoisomerase I (100 kd) catalyzes the relaxation of supercoiled DNA by cleaving a single phosphodiester bond to form a temporary phosphoryl tyrosine diester. This intermediate is called the xe2x80x9ccleavable complex.xe2x80x9d The other end of the cleaved strand is free, and can xe2x80x9cunwindxe2x80x9d before the DNA chain is resealed by reverse of the original reaction. Topoisomerase I acts without cofactors, its reactions are fully reversible, and it is thought to be especially important for unwinding DNA (thermodynamically favorable) during replication. In contrast, topoisomerase II acts by cleaving and resealing (after strand passage) both strands of DNA, and its reactions are coupled with ATP hydrolysis.
There is now very strong evidence that camptothecin kills cells by binding to and stabilizing the covalent DNA-topoisomerase I complex in which one strand of DNA is broken (the cleavable complex). The progression from the ternary camptothecin/topoisomerase I/DNA complex to cell death is not well understood, and is the subject of intense investigation. Several lines of evidence (including the complete reversibility of ternary complex formation) indicate that the ternary complex does not simply tie up DNA, but itself actively initiates cell death. For this reason, camptothecin is often called a xe2x80x9ctopoisomerase poison.xe2x80x9d
Until very recently, camptothecin and its close relatives were the only known topoisomerase I poisons. In contrast, there are now many known antitumor agents that are topoisomerase II poisons. These include large classes of intercalators like the acridines and anthracyclines that were originally thought to interact only with DNA. Such topoisomerase II poisons may be inherently less selective than camptothecin because their interactions with DNA do not require topoisomerase II. Important non-intercalative topoisomerase II poisons include members of the podophyllotoxin class.
Camptothecin is being touted as an unusually important lead in cancer chemotherapy because of its selectivity. The (potential) selective toxicity of camptothecin towards cancer cells emanates from two sources: 1) camptothecin is highly selective for the DNA/topoisomerase I cleavable complex, and 2) replicating cancer cells contain elevated levels of topoisomerase I (15-fold increases over normal cells have recently been measured).
New interest resulting from the identification of camptothecin""s mechanism of action has initiated (i) structure-activity relationship studies and (ii) a new series of clinical trials. Recent tests in xenografts by Potmesil and coworkers were very promising. See Giovanella, B. C., et al., Science, 246, 1046 (1989). Racemic 9-aminocamptothecin was found to be very effective in treating mice carrying colon cancer xenografts. Indeed most of the mice in the study were cured by 9-aminocamptothecin at dose levels that were well tolerated. The improved efficacy of 9-aminocamptothecin compared to current drugs used in colon cancer chemotherapy (like 5-fluorouracil) was dramatic. 10,11-Methylenedioxycamptothecin also showed very good promise. The significance of these results is very high. Human colon cancer is a major problem in clinical oncology, and one in twenty-five Americans will develop this disease during their lifetime.
More recently, other close relatives (analogs) of camptothecin have also emerged as excellent candidates for chemotherapy against a variety of tumor types. In an attempt to overcome the problem of low solubility encountered earlier with camptothecin, most of such compounds are designed to be water-soluble. Several of these compounds are undergoing clinical trials. See Sinha, B. K., Drugs, 49, 11 (1995). Pommier, Y. et al., J. Natl. Cancer Inst., 86, 836 (1994). Potmesil, M. Cancer Res., 54, 1431 (1994). Curran, D. P., xe2x80x9cThe Camptothecins: A Reborn Family of Antitumor Agents,xe2x80x9d J. of the Chinese Chem. Soc., 40, 1-6 (1993), the disclosure of which is incorporated herein by reference. See also Sawada, S., Chem. Pharm. Bull., 39, 1446 (1991); Giovanella, B. C., et al., Science (Washington, D.C.), 246, 1046 (1989); Kingsbury, W. D., et al.; Med. Chem., 34, 98 (1991); Sawada, S., et al.; Chem. Pharm. Bull., 39, 1446 (1991), Nicholas, A. W., et al. J. Med. Chem. 33, 972 (1991).
Such compounds, for example, include topotecan (often called TPT), currently undergoing phase III studies, and irinotecan (often called CPT-11), currently undergoing phase II studies. See Abigerges, D., et al., J. Clin. Oncol., 13, 210 (1995). Potmesil, M., Cancer Res., 54, 1431 (1994). Miller, A. et al., J. Clin. Oncol., 12, 2743 (1994). Fukuoka, M. et al., Canc. Chemotherap. Pharmacol., 34, 105 (1994). Shimada, Y. et al., J. Clin. Oncol., 11, 909 (1993). Another analog, 10,11-ethylenedioxy-7-(4-methylpyrazino)-camptothecin, has been recently introduced by Glaxo and is now in clinical trials. See Eur. Pat. Appl. EP 540,099 (C1. C07D491/22), May 5, 1993, U.S. application Ser. No. 784,275, Oct. 29, 1991. Luzzio, M. J. et al., J. Med. Chem., 38, 395 (1995). See also Wall, M. E. et al., J. Med. Chem., 36, 2689 (1993). The results of these recent trials suggest that these compounds hold excellent promise for the clinical treatment of a number of types of refractory solid tumors.
Moreover, recent trials using new formulations have recently opened new opportunities for camptothecin derivatives previously dismissed for their poor water solubility. In this regard, it has been discovered that (i) (S)-camptothecin itself can be formulated in 20% interlipid, and (ii) this formulation is active both intramuscularly and orally. These treatments were found far superior to previous intravenous treatments. With this formulation, non-toxic doses of camptothecin suppressed growth and induced regression of cancer in thirteen human xenograft lines, including colon, lung, breast, stomach, ovary, and malignant melanoma. Camptothecin was more effective than any other clinical drug tested. See Giovanella, B. C. et al., Cancer Res., 51, 3052 (1991).
More recently liposome-incorporated camptothecin has shown, when administered intramuscularly, excellent anti-tumor activities on mice xenografted with human malignant melanoma and breast carcinoma. This mode of administration appears to hold very good promise, particularly in the treatment of human lymph node metastases. See Giovanella, B. C., Anti-Cancer Drugs, 6, 83 (1995).
The large variety of camptothecin derivatives or analogs synthesized and studied has allowed study of the features of the molecule which are preferable for cytotoxicity. See Kaufmann, S. H. et al., J. Natl. Cancer Inst., 85, 271 (1993) and references cited therein; Uehling, D. E. et al., J. Med. Chem., 38, 1106 (1995); Luzzio, M. J. et al., J. Med. Chem., 38, 395 (1995); Wang, H. K. et al., Bioorg. Med. Chem. Lett., 5, 77 (1995); Sawada, S. et al., Chem. Pharm. Bull., 42, 2518 (1994); Terasawa, H. et al., Heterocycles, 38, 81 (1994); Terasawa, H. et al., J. Med. Chem., 37, 3033 (1994); Wall, M. E. et al., J. Med. Chem., 36, 2689 (1993).
A number of structural features identified by those studies and believed important for activity are briefly summarized below. It is believed that only the (S) enantiomer of camptothecin is responsible for its bioactivity. The (R) enantiomer is believed to be inactive. The closed lactone ring and its xcex1-hydroxyl group in position 20 are believed to be important for bioactivity. Substitution or modification of camptothecin rings reveal that numerous and varied substitution of its xe2x80x9cnorthernxe2x80x9d and xe2x80x9cwesternxe2x80x9d regions, particularly the A- and B-rings, are compatible with the biological activity. Thus, positions 7, 9 and 10, and particularly position 7, have been widely substituted with hydrophilic and lipophilic groups such as alkyl, alkylamine, benzyl, hydroxy, amino and halo groups, to provide camptothecin derivatives retaining biological activity. These groups may be unsubstituted or substituted. Such compounds include the water-soluble topotecan and irinotecan. In general, it appears that substantially any substitution can be made at the 7, 9 and 10 positions while maintaining activity. Other tolerated modifications of the A- and B-ring are the replacement of one aromatic carbon with a nitrogen atom, such as 7- or 11-azacamptothecin which exhibited increased water-solubility while maintaining activity.
It is believed that substitutions possible at position 11 to retain biological activity may be more limited. However, such substitutions include the recently developed 10,11-ethylenedioxy-7-(4-methylpyrazinomethyl)-camptothecin of Glaxo and several new 11-fluoro camptothecin analogues recently disclosed by Sawada.
In contrast, for the C-ring, the only acceptable modifications discovered to date are those in position 5. Moreover, it is believed that biological activity will be retained only if the planarity of the molecule is conserved. Concerning the ring E, the ethyl group of camptothecin has been replaced by allyl, propargyl, and benzyl groups without significantly reducing the activity.
In addition to the interest in camptothecin and its analogs as anti-tumor agents, the interest in such compounds has recently increased to even greater levels upon the discovery that camptothecin is a potent antiretroviral agent. Preil and coworkers showed that camptothecin and analogs: 1) inhibited retroviral topoisomerase I, 2) prevented retroviral infections in healthy cells, 3) reduced and eliminated retroviral infections and infected cells, and 4) did not harm cells at useful dose levels. See Priel, E., et al., AIDS Res. Hum. Retroviruses 7, 65 (1991). Topoisomerase II inhibitors were ineffective. These results suggest that camptothecin may represent a new avenue of investigation for the potential treatment of AIDS.
Camptothecin was synthesized about ten times during the 1970s, although some later syntheses are modifications of earlier ones. Syntheses based on the Friedlander quinoline synthesis to construct ring B were most common. Ejima, A., et al., J. Chem. Soc., Perkin Trans. 1, 27 (1990); Earl, R. E. and Vollhardt, K. P. C., J. Org. Chem. 1984, 49, 4786; Ihara, M. et al., J. Org. Chem., 48, 3150 (1983); Cai, J. C. and Hutchinson, C. R., Chem. Heterocycl. Compd. 25, 753 (1983); Hutchinson, C. R., Tetrahedron 37, 1047 (1981); Cai, J. C. and Hutchinson, C. R., The Alkaloids: Chemistry and Pharmacology; Brossi, A. Ed.; Academic Press: New York, Vol. 21, p. 101 (1983); Schultz, A. G., Chem. Rev. 73, 385 (1973). Many syntheses are racemic, but resolutions have been reported. See Wani, M. C., et al. J. Med. Chem., 30, 2317 (1987). More recently, a chiral auxiliary approach to asymmetric ethylation was described. See Ejima, A., et al., Tetrahedron Lett., 30, 2639 (1989).
New or improved synthetic routes to camptothecin have recently been described by a number of laboratories. See Comins, D. L.; Hong, H.; Jianhua, G. Tetrahedron Lett., 35, 5331 (1994). Comins, D. L.; Hong, H.; Saha, J. K.; Gao, J. H. J. Org. Chem., 59, 5120 (1994). Fang, F. G.; Xie, S. P.; Lowery, M. W. J. Org. Chem., 59, 6142 (1994). Rao, A. V. R.; Yadav, J. S.; Muralikrishna, V. Tetrahedron Lett., 35, 3613 (1994). Wang, S.; Coburn, C. A.; Bornmann, W. G.; Danishefsky, S. J. J. Org. Chem., 58,611 (1993).
It remains very desirable, however, to develop short, practical syntheses of camptothecin and its analogs.
Accordingly, the present invention provides a short, convergent total synthesis of camptothecin and related compounds via a novel 4+1 radical annulation.
In one embodiment, the 4+1 radical annulation is followed by another cyclization to simultaneously assemble rings B and C of camptothecin and related compounds. In that regard, the present invention provides generally a method of synthesizing tetracyclic compounds having the general formula 
which are intermediates in many syntheses of (xc2x1)-camptothecin and related compounds. The conversion of these intermediates to (xc2x1)-camptothecin and related compounds is accomplished in two steps as known in the art: hydroxymethylation and oxidation.
The synthesis of the tetracyclic intermediates comprises the step of a 4+1 radical annulation wherein the following novel precursor: 
is reacted with an aryl isonitrile such as phenyl isonitrile. Y is preferably selected from the group consisting of xe2x80x94N and xe2x80x94CR3. The aryl isonitrile may be unsubstituted, monosubstituted, disubstituted or trisubstituted.
R1, R2, R3 and R6 are preferably independently selected from the following groups: hydrogen, a normal alkyl group, a branched alkyl group, an allyl group, a benzyl group, an alkynyl group, a propargyl group, an alkoxyl group, a halo group, a silyl group, an amino group, a cyano group, and an acyl group. R6 is more preferably selected from an alkoxyl group or a halo group. Moreover, the groups set forth for R1, R2, R3 and R6 may be substituted with a wide variety of substituents to synthesize camptothecin analogs retaining activity.
For example, normal (unbranched) and branched alkyl groups, which preferably range between C1 and C15 (and more preferably between C1 and C10), may preferably be substituted with a group or groups including, but not limited to, a benzyl group, a phenyl group, an alkoxyl group, a hydroxy group, an amino group (including, for example, free amino groups, alkyl amino groups and aryl amino groups) and an acyloxy group. Alkynyl groups (xe2x80x94Cxe2x95x90Cxe2x80x94R) may preferably be substituted with a group or groups including, but not limited to, an alkoxyalkyl group, an amino alkyl group and a benzyl group. Acyl groups (RCOxe2x80x94) may preferably be substituted with a group of groups including, but not limited to, a branched or unbranched allyl group (preferably between C1 and C15; more preferably between C1 and C10), a haloalkyl group (for example, a perfluoroalkyl group), an alkoxyl group, an amino group an a hydroxy group. Alkoxyl groups (xe2x80x94OR) preferably comprise a branched or unbranched alkyl group (preferably between C1 and C15; more preferably between C1 and C10). Amino groups (xe2x80x94NRaRbRc) may preferably be substituted with a group or groups including, but not limited to, hydrogen, an acyl group, a branched or unbranched alkyl group (preferably between C1 and C15; more preferably between C1 and C10) an alkoxyl group and an aryl group (preferably a phenyl or napthyl group). Silyl groups may preferably be substituted with a group or groups including, but not limited to, a branched or unbranched alkyl group (preferably between C1 and C15; more preferably between C1 and C10) and an aryl group (preferably a phenyl or napthyl group; more preferably a phenyl group).
In general, R1, R2, R3 and R6 are preferably not excessively bulky to maintain activity of the resultant camptothecin analog. Preferably, therefore, R1, R2, R3 and R6 independently have a molecular weight less than approximately 250. More preferably R1, R2, R3 and R6 independently have a molecular weight less than approximately 200.
R4 is preferably selected from a primary or secondary alkyl group (preferably between C1 and C15; more preferably between C1 and C10), an allyl group, a propargyl group and a benzyl group. More preferably, R4 is an ethyl group. R5 is preferably selected from a linear or branched alkyl group (preferably between C1 and C15; more preferably between C1 and C10) or a benzyl group. Most preferably, R5 is selected from linear or branched alkyl groups in the range of C1 to C6.
The above-described synthetic scheme is very useful for large-scale production of racemic camptothecin and analogs thereof.
In another embodiment, the present invention provides a variant of the above 4+1 radical annulation/cyclization scheme in which the key E-ring lactone is preferably introduced in optically active form prior to the radical annulation/cyclization.
In that regard, the present invention provides short, convergent, and practical syntheses of optically active pentacyclic compounds having the general formula 
Included among these compounds, for example, are (20S)-camptothecin itself, and the related anti-tumor agents (20S)-topotecan, (20S)-irinotecan and (20S)-10,11-ethylenedioxy-camptothecin as follows.
The synthesis of the pentacyclic compounds comprises the step of a 4+1 radical annulation/cyclization wherein the following novel bicyclic precursor: 
is reacted with an aryl isonitrile such as phenyl isonitrile.
In the case of a triple bond, Y is preferably selected from the group consisting of xe2x80x94N and xe2x80x94CR3. In the case of a double bond, Y is preferably xe2x80x94CH2 or xe2x80x94CHR3. X is preferably I or Br. The aryl isonitrile may be unsubstituted, monosubstituted, disubstituted or trisubstituted.
R1, R2, R3 and R6 are preferably selected independently from the groups described previously for the synthetic route to racemic camptothecin and analogs thereof. R4 is preferably selected from a primary or secondary alkyl group (preferably between C1 and C15; more preferably between C1 and C10), an allyl group, a propargyl group and a benzyl group. Most preferably R4 is an ethyl group.
This synthetic route is thus useful for the large scale production of (20S)-camptothecin and its analogs, including, but not limited to (20S)-topotecan and (20S)-irinotecan. The synthetic routes of the present invention allow rapid, simple and controlled substitution in the camptothecin series of compounds while maintaining activity.
The present invention also provides the following novel precursor 
wherein R9 is preferably an alkyl group (branched or unbranched and preferably between C1 and C15; more preferably between C1 and C10). R10 is preferably selected from the group consisting of xe2x80x94CHO and xe2x80x94CH2OCH2CHxe2x95x90CHR11, wherein R11 is selected from the group consisting of an alkyl group (branched or unbranched and preferably between C1 and C15; more preferably between C1 and C10), a vinyl group, an ethynyl group and a phenyl group. R12, R13 and R14 are preferably independently selected from an alkyl group (branched or unbranched and preferably between C1 and C15; more preferably between C1 and C10) and an aryl group (preferably a phenyl group or a napthyl group; more preferably a phenyl group). X is preferably selected from the group consisting of I and Br.
Still further, the present invention provides the following novel precursor 
wherein R4, R9, R12, R13 and R14 are preferably selected as set forth above.